Molecular mechanisms of vascular calcification and their connection to coronary disease risk

血管钙化的分子机制及其与冠心病风险的关系

• 批准号: 10673742
• 负责人: THOMAS QUERTERMOUS
• 金额: $ 58.92万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10673742
• 关键词: ATAC-seq; Address; Anatomy; Animals; ApoE knockout mouse; Apolipoprotein E; Aryl Hydrocarbon Receptor; Atherosclerosis; Binding; Blood Vessels; Cardiovascular Diseases; Cell Lineage; Cell model; Cells; Cellular Assay; Cellular Morphology; Chromatin; Chromosome Mapping; Complex; Coronary Arteriosclerosis; Coronary artery; Coronary heart disease; Development; Developmental Process; Disease; Enhancers; Epigenetic Process; Fibroblasts; Functional disorder; Gene Expression; Genes; Genetic Risk; Genetic Transcription; Goals; Human; Human Genome; In Vitro; Investigation; Knock-out; Knockout Mice; Lesion; Link; MADH3 gene; Maps; Medial; Mediating; Modeling; Molecular; Morphology; Mus; Osteogenesis; Pathway interactions; Phenotype; Process; Regulation; Research; Risk Assessment; Role; Scientist; Signal Pathway; Signal Transduction; Signaling Molecule; Smooth Muscle Myocytes; Subcellular Anatomy; TGFB1 gene; Therapeutic; Tissues; Transposase; Vascular Diseases; Vascular Smooth Muscle; Vascular calcification; Wild Type Mouse; Work; blocking factor; calcification; cell type; disorder risk; genome wide association study; genome-wide; in vivo; mouse model; novel; programs; public health relevance; recruit; single-cell RNA sequencing; transcription factor; transcription factor USF; transcriptome sequencing

The combination of lineage tracing and single cell RNA sequencing (scRNAseq) in mouse atherosclerosis models has created a paradigm shift in our understanding of vascular disease, showing that lesion smooth muscle cells (SMC) undergo phenotypic transitions into derivative cells with multiple complex phenotypes. We identified TCF21 as a coronary artery disease (CAD) associated gene mapped by genome-wide association studies (GWAS) and showed that this gene regulates a disease-related transition of SMC to a fibroblast like phenotype, producing cells we term “fibromyocytes.” Further, we and others have shown that medial SMC can also transition to a second SMC-derived cellular phenotype, characterized by expression of genes known for their role in endochondral bone formation, substantiating and expanding previous work investigating this process that is linked to intimal vascular calcification. We showed that this chondrogenic process, which gives rise to cells we term “chondromyocytes” (CMC), is actively inhibited by two CAD associated genes, one encoding the TGFB1 signaling molecule SMAD3, and the other encoding the environmental sensing aryl hydrocarbon receptor (AHR). Knockout (KO) of both genes in mouse models showed increased transition to CMC, larger lesion size and increased vascular calcification. These studies identified SOX9 as a primary driver of the phenotypic transition to the CMC phenotype. Our longterm goal is to elucidate the molecular mechanisms that mediate the detrimental CMC transition. Our Central Hypothesis postulates that SOX9 is a key initiator of this chondrogenic process in the vascular wall, as it is in endochondral bone formation, and regulation of its expression and function in SMC is intimately linked to vascular calcification and disease risk. Our objective is thus to determine the upstream epigenetic signals that modulate SOX9 expression, and how SOX9 expression contributes to CMC development and vascular calcification. Specifically, in Aim 1 we will employ Sox9 KO and SMC lineage tracing in the ApoE KO mouse atherosclerosis model to characterize the effect of this gene on SMC cell state transitions, and the impact of perturbing these transitions on disease morphology and cellular anatomy. In Aim 2, we will conduct scRNAseq in these mice to characterize the SMC gene expression program downstream of Sox9 in this cell type. Single cell assay of transposase accessible chromatin sequencing (scATACseq) in the same animals will map enhancers genome-wide that are differentially regulated in CMC phenotypic transition, and identify specific transcription factors (TFs) that bind these enhancers to regulate expression of CMC genes. Studies proposed in Aim 3 will employ in vitro studies to characterize the transcriptional and epigenetic mechanism by which SOX9 interacts with the inhibitory factors SMAD3 and AHR, and novel TFs that promote transition to the CMC phenotype. The proposed studies will identify cellular and molecular mechanisms that mediate SMC transition to CMC, and the relationship of this process to vascular calcification and disease risk.

谱系追踪和单细胞RNA测序（scRNAseq）在小鼠动脉粥样硬化中的联合应用 模型在我们对血管疾病的理解中创造了一个范式转变，表明病变平滑 肌细胞（SMC）经历表型转变为具有多种复杂表型的衍生细胞。我们 TCF 21被鉴定为通过全基因组关联定位的冠状动脉疾病（CAD）相关基因 研究（GWAS），并表明该基因调节SMC向成纤维细胞样的疾病相关转变， 表型，生产细胞，我们称之为“纤维肌细胞”。此外，我们和其他人已经表明，中膜SMC可以 也转变为第二SMC衍生的细胞表型，其特征在于已知的 它们在软骨内骨形成中的作用，证实和扩展了以前的研究工作， 与血管内膜钙化有关的过程。我们证明了这个软骨形成过程，这使得 上升到细胞，我们称之为“软骨肌细胞”（CMC），是积极抑制两个CAD相关基因，一个 一个编码TGFB1信号分子SMAD3，另一个编码环境传感芳基 碳氢化合物受体（AHR）。在小鼠模型中，两种基因的敲除（KO）均显示出增加的向 CMC、病变尺寸增大和血管钙化增加。这些研究将SOX 9确定为主要驱动因素 从表型转变为CMC表型。我们的长期目标是阐明 介导有害CMC转变的机制。我们的中心假设假定SOX 9是 与软骨内骨一样，它是血管壁软骨形成过程的关键启动子 平滑肌细胞的形成及其表达和功能的调节与血管内皮细胞的生长密切相关。 钙化和疾病风险。因此，我们的目标是确定上游表观遗传信号， 调节SOX 9表达，以及SOX 9表达如何促进CMC发育和血管形成。 钙化具体而言，在目标1中，我们将在ApoE KO小鼠中采用Sox 9 KO和SMC谱系追踪 动脉粥样硬化模型，以表征该基因对SMC细胞状态转换的影响，以及 扰乱疾病形态和细胞解剖学上的这些转变。在目标2中，我们将进行scRNAseq 在这些小鼠中表征该细胞类型中Sox 9下游的SMC基因表达程序。单个 将在相同动物中进行转座酶可及染色质测序（scATACseq）的细胞测定， 在CMC表型转变中差异调节的全基因组增强子，并鉴定特异性 这些增强子与转录因子（TF）结合以调节CMC基因的表达。拟议的研究 目的3将采用体外研究来表征转录和表观遗传机制， SOX 9与抑制因子SMAD 3和AHR以及促进向CMC转变的新型TF相互作用 表型拟议的研究将确定介导SMC转变的细胞和分子机制 CMC，以及该过程与血管钙化和疾病风险的关系。